Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an anion transporter of chloride and bicarbonate. Defective or deficient CFTR leads to severe mucoobstructive lung disease and severe morbidity and early mortality due to lung infections. Even though substantial advances in our understanding of CF pathogenesis have been made, we still do not fully understand the pathophysiologic mechanisms that lead to decreased mucociliary clearance (MCC) in CF patients. Two critical barriers underlie this roadblock: 1) there have been no tools available for visualizing the important microstructural, functional, and biomechanical features of the respiratory mucosa and mucus in vivo, and 2) it has been difficult to study young CF patients to determine the fundamental defects of this disease, prior to the occurrence of secondary phenomena such as infection and inflammation that confound the study of CF pathogenesis. In this grant, we will overcome these barriers through a novel cross-sectional optical microscopy technology termed 1-?m OCT (?OCT), that has been shown in preliminary studies to clearly visualize the structural and functional microanatomy of bronchial epithelial cells and trachea ex vivo. With ?OCT, we have been able to simultaneously and quantitatively monitor airway surface liquid (ASL) and periciliary layer (PCL) depths, ciliary beating, and mucociliary transport while also measuring mucus viscosity by native particle tracking techniques. We propose to advance this technology by building an improved imaging system and a novel pulmonary catheter that will enable ?OCT to be used in the airways of living human subjects. In addition, potential difference (PD) electrodes will be integrated within the sheath of the ?OCT catheter, enabling co-localized measurements of CFTR ion channel activity. This device will then be employed to investigate the airways of young children with CF, prior to the onset of structural lung disease. Our experiments are intended to define the earliest events that initiate CF pathogenesis, including the relationship of ASL regulation to mucociliary transport (MCT) and mucus biogenesis while also determining the roles of chloride and bicarbonate transport towards regulating these pathways. By accomplishing these objectives, we will be able to address key hypotheses and resolve controversies in the field regarding the interrelationships between the CFTR ion transport defect and the regulations of ASL/PCL depth, ciliary function, and the physical properties of mucus, and the impact of novel CFTR modulators on these pathways. The end product of this research will expand the knowledgebase regarding the fundamental pathophysiology of CF, resulting in new avenues for research and development of pharmacologic agents that specifically target ion transport, mucus biogenesis, or other primary pathways underlying CF pathogenesis. This work will also enable imaging of the functional microanatomy of the lung in living human patients in the future, which will significantly impact those with CF by providing an imaging modality for assessing the progression of CF lung disease and the efficacy of drugs administered to combat mucus stasis. (End of Abstract)